The Myelodysplastic Syndrome (MDS) is predominantly a disease of older age, and with the aging of the population, an increase in cases is expected. Our proposal is aimed at improving our understanding of the pathophysiology of MDS, and to identify cellular or molecular targets suitable for novel treatment strategies. MDS is a clonal disorder of hematopoietic stem cells; however, there is evidence that factors in the microenvironment contribute to the propagation of the disease. Thus, we propose to characterize interactions between the marrow microenvironment and hematopoietic precursors and identify signaling pathways that determine programmed cell death (apoptosis) and clonal survival. Specifically, we will 1) define stroma-dependent activities that affect survival/expansion of clonal and non-clonal hematopoietic precursors from MDS marrow. In an in vitro system using myeloid cell lines and primary MDS cells, we will characterize the functional relevance of tumor necrosis factor (TNF)alpha-induced alterations in gene expression in stroma for the support of clonal or non-clonal hematopoietic precursors (TNFalpha is upregulated in MDS). In a xenogeneic in vivo model we will determine the role of human stroma for the survival of MDS-derived clones transplanted into mice. We will 2) characterize apoptotic and proliferative events in MDS marrow and correlate these with disease progression. We will define interactions of TNFalpha-initiated signals that control apoptosis and proliferation, in particular, the roles of NFkappaB and the anti-apoptotic molecule, FLIP. Preliminary in vitro data show that overexpression of FLIP enhances cell survival. We will now determine in vivo whether genetic modification of FLIP expression in MDS cells transplanted in immunodeficient mice affects survival and allows for expansion of the clone. The techniques involved in pursuit of our objectives include in vitro assays of hematopoiesis, apoptosis, and proliferation in in vitro culture systems. The relevance of in vitro findings will be validated in vivo in a xenogeneic transplant model of cell lines and primary MDS cells in immunodeficient mice in which propagation of normal and clonal cells will be characterized. By using interventions that block relevant signals or by genetically modifying molecules that play pivotal roles, we will identify potential targets for novel therapeutic strategies. The human population is aging, and the incidence of MDS increases with age. Novel treatment approaches are needed that are tolerated by older individuals and, hopefully, will not interfere with their quality of life. To achieve these objectives, we must better understand the underlying mechanisms of MDS and thereby identify ways by which the disease process can be arrested, progression prevented, and the disease hopefully be cured.